The Experimental Study On The Neurological Mechanisms Of The Prostatic Inflammation Pain

Background and Objective Chronic prostatitis(CP) is a very extremely common urologic disease. Pain appears to be the most prominent clinical manifestation of chronic prostatitis and this symptom significantly reduced the quality of life and functional status of those individuals. But the neurological mechanisms of its occure and development are unclear. Pain of the chronic prostatitis manifest characteristics of typical visceral referred pain and usually accompanied by voiding dysfunction and pelvic floor dysfunction. Pain is a kind of neurological event. The frequent association of the pain of the chronic prostatitis and the voiding dysfunction and pelvic floor dysfunction should have neurological basis. In addition to being an intracellular energy source, ATP is an important neurotransmitter or neuromodulator. It depolarizes DRG neurons by eliciting fast-and slow-inactivating inward currents mediated by P2X receptors. P2X3 expressed selectively in nociceptive medial-sized and small-sized neurons of the DRGs. Insults to afferent fibers and peripheral tissues ,such as neuropathy and inflammation frequently give rise to exaggerated responses to non-noxious and noxious stimuli(allodynia and hyperalgesia). These pathological responses are thought to arise from sensitization of P2X receptor-mediated responses in DRG and dorsal horn neurons to external stimuli. It has not been reported about the changes of excitability of P2X receptor-mediated DRG neurons and expression of P2X3 receptor in the DRG neurons during the pain of CP. The most recent studies show, activition of glial cells(mainly astroglia and microglia) in spinal cord can lead to hyperalgesia and persist pain status. The pain of CP often show characteristics of chronic persist pain and hyperalgesia. It should have some relationships with the activation of the astroglia and microglia in the spinal cord. But to our knowledge, there is no report about the relationship between them. The objective of this study is to investigate the neurological basis and neuropathological process of the refered pain of CP , so as to provide new therapeutic targets and experimental basis for clinical treatment. Material and Methods The distribution of plasma extravasation using Evans blue dye was determined after capsaicin irritation of the prostate and bladder of the rats, and the distribution of dye extravasation was analyzed. Substance P(SP) expression within the spinal cord was determined using hybridization in situ method after formalin irritation of the prostate, bladder and tail root. The changes of ATP-evoked responses and the expression and distribution of P2X3 receptor in the DRG neurons in the L6-S1 spinal cord were investigated using whole-cell patch clamp techniques, Western blot and fluorescent immunohistochemical staining after Complete Freund′s adjuvant(CFA) irritation of the prostate of the rats at different times. The activation of astroglia and microglia in the spinal cord were investigated using immunohistochemical staining after CFA irritation of the prostate of the rats at different times. Results 1. Capsaicin irritation of the prostate resulted in plasma extravasation in L5 to S2 dermatomes(primarily in L6 and S1).In rats receiving bladder irritation,the distribution of plasma extravasation showed a similar pattern to that observed in animals receiving prostatic irritation. 2. Formalin irritation of the prostate,bladder and tail root resulted in expression of SP within the lumbosacral spinal cord. With each treatment the majority of positive SP were in the L6 and S1 segments. In all three groups the highest percentages of positive SP were observed in deeper laminae,including the dorsal commissure and sacral parasympathetic nucleus. 3. Obvious inflammation were observed in the prostate after CFA irritation at the third day and tenth day, and expression of SP were also observed in the DRG neurons in the L6 and S1 spinal cord. 4. Morphology of DRG with distinct soma diameter of 41-60μm,30-40μm,13-29μm and single or double dentrite were readily distinguished,and were not changed after CFA irritation of the prostate. 5. There is no significant changes and differences of resting membrane potential,series resistance , membrane capacity of DRG neurons of control group , inflammation group(3d)and inflammation group(10d),using whole-cell patch clamp method. That is to say passive electrophysiological characteristics of DRG neurons were not markedly changed after CFA irritation of the prostate.Action potential (AP)was recorded when DRG neurons were stimulated by a series of current from hyperpolarization to depolarization(-60pA-140Pa,step=20Pa,time=200ms). The threshold and time of AP were significantly changed after CFA irritation of prostate at 10 days(p<0.01). However,the threshold and time of AP were not markedly changed after CFA irritation of prostate at 3 days. 6. Membrane potential of DRG neurons fluctuates spontaneously under current clamp. Membrane potential of neurons of control and inflammation(3d)group were under threshold potential,and all recorded neurons have not action potential. However,10.26%(4/39)of neurons showed spontaneous action potential at membrane potential after CFA irritation of prostate at 10 days. 7. Rapid application of ATP to DRG neurons evoked action potential under current clamp. The response to ATP was blocked by suramin. Different concentrations of 0.1μM,1μM,10μM and 100μM lead to depolarization of membrane potential of DRG neurons under current clamp holding DRG neurons potential about –60mV. But the depolarization amplitude of acton potential of different groups(control group vs inflammation(10d)group) evoked by the same ATP concentrations and of the same group evoked by different ATP concentrations were significantly different(p<0.01). Median effective concentration(EC50)of ATP of control and inflammation(10d)group were 4.26±1.69μM and 1.10±0.90μM respectively(p<0.01). 8. Rapid application of different ATP concentrations (0.1μM,1μM,10μM and 100μM) to DRG neurons evoked a fast-activating inward current under voltage clamp holding DRG neurons potential about –60mV. Amplitudes of inward current of control group and inflammation(10d)group evoked by the same ATP concentration,and of the same group of control or inflammation(10d) group evoked by different ATP concentrations were markedly different(p<0.01).EC50 of ATP of control and inflammation(10d) group were 4.07±1.38μM and 1.06±0.87μM respectively(p<0.01). In control and inflammation(10d)group,32%(8/25)and 36%(9/25)of neurons tested responded to ATP with transient current,and 54.17%(13/24)and 52%(13/25)of neurons with a sustained current respectively. Percentage of transient current and sustained current were not markedly different after CFA irritation of the prostate(p>0.05). 9. Expression of P2X3 receptor increased significantly after 10 days of CFA irritationof the prostate tested by methods of western blot and fluorescent immunohistochemical staining,and P2X3 selectively expressed in the middle-sized and small-sized nociceptive DRG neurons. 10. Activation of astrocyte and microglia was significantly increased in deeper laminae and superficial laminae of the dorsal horn of L6-S1 spinal cord at the third day, tenth day and twenty-eighth day after CFA irritation of the prostate, and the most obvious activation occurred at the tenth day. Conclusions 1. The referred pain status in inflammation of the prostate and bladder is neurogenically mediated. There should be significant overlaps of nociceptive neurons in the L6 and S1 segments of the spinal cord, which receive nociceptive inputs from pelvic soma(such as pelvic floor muscles) and viscera(such as the prostate and bladder). 2. P2X3 receptor of DRG neurons were not markedly changed after CFA irritation of the prostate at 3days; but expression of P2X receptor of DRG neurons was markedly enhanced, and sensitivity and affinity of P2X receptor channel were markedly reinforced after CFA irritation of the prostate at 10 days. These results suggest that increase of expression and sensitivity of P2X receptor may have important roles in the development and maintain of the pain of prostatic inflammation. 3. CFA irritation of the prostate resulted in obvious activation of astroglia and microglia in the L6 and S1 segments of the spinal cord, and the activation significantly increased after 3days, 10days and 28days. These results suggest that activation of glia in the spinal cord may play an important role in the neuropathological process of the pain of prostatic inflammation. The molecular mechanisms should be further explored.